Inhibition Of Epigenetic Regulators Research Articles

Structure-guided design of a selective inhibitor of the methyltransferase KMT9 with cellular activity.

Inhibition of epigenetic regulators by small molecules is an attractive strategy for cancer treatment. Recently, we characterised the role of lysine methyltransferase 9 (KMT9) in prostate, lung, and colon cancer. Our observation that the enzymatic activity was required for tumour cell proliferation identified KMT9 as a potential therapeutic target. Here, we report the development of a potent and selective KMT9 inhibitor (compound 4, KMI169) with cellular activity through structure-based drug design. KMI169 functions as a bi-substrate inhibitor targeting the SAM and substrate binding pockets of KMT9 and exhibits high potency, selectivity, and cellular target engagement. KMT9 inhibition selectively downregulates target genes involved incell cycle regulation and impairs proliferation of tumours cells including castration- and enzalutamide-resistant prostate cancer cells. KMI169 represents a valuable tool to probe cellular KMT9 functions and paves the way for the development of clinical candidate inhibitors as therapeutic options to treat malignancies such as therapy-resistant prostate cancer.

P04.04.A GENOME-WIDE CRISPR/CAS9 KNOCKOUT SCREENS REVEAL EPIGENETIC REGULATORS AS DRUGGABLE TARGETS FOR SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract BACKGROUND Sonic hedgehog medulloblastoma (SHH-MB) is a malignant pediatric brain tumor that accounts for 30% of all MBs and is characterized by constitutive activation of the SHH signaling pathway. Besides the standard-of-care therapies, current therapies are targeting Smoothened (SMO) receptor, in order to inhibit the pathway. However, acquired or de novo resistance against SMO inhibition limits its efficacy. Therefore, our aim is to unravel novel therapeutic targets for SHH-MB, potentially beyond the SHH pathway. MATERIAL AND METHODS We performed genome-wide CRISPR/Cas9 knockout screens, in order to decipher cancer dependencies and synthetic lethal interactors for SHH-MB. The hits identified were first validated genetically and pharmacologically in in vitro assays, while further molecular analyses including RNAseq and differential methylation were also conducted. Moreover, the identified hits were evaluated in in vivo mouse models representing normal cerebellar, as well as SHH-MB development. RESULTS Functional genomics identified members of the epigenetic machinery, such as Dnmt1 and Smarca5 as context-specific dependencies for SHH-MB. We prove that DNMT1 inhibition is efficacious against SMO-resistant and sensitive SHH-MB cell lines, by inhibiting SHH pathway output, as shown via RNAseq. A further knockout drug screen unraveled novel synthetic lethal interactors for DNMT1 inhibitors. Genetic ablation and pharmacological inhibition of epigenetic regulators prolongs survival of established mouse models of SHH-MB. CONCLUSION Summarizing, our data indicate inhibitors of epigenetic regulators as novel therapeutic targets for SHH-MB irrespective of mutational drivers within the SHH pathway. We further propose combinatorial treatment approaches for this tumor entity that involve DNMT1 inhibitors.

Crucial Role of Lysine-Specific Histone Demethylase 1 in RANKL-Mediated Osteoclast Differentiation.

Epigenetic regulators are involved in osteoclast differentiation. This study proposes that the inhibitors of epigenetic regulators could be effective in the treatment of osteoporosis. This study identified GSK2879552, a lysine-specific histone demethylase 1 (LSD1) inhibitor, as a candidate for the treatment of osteoporosis from epigenetic modulator inhibitors. We investigate the function of LSD1 during RANKL-induced osteoclast formation. LSD1 small-molecule inhibitors effectively inhibit the RANKL-induced osteoclast differentiation in a dose-dependent manner. LSD1 gene knockout in macrophage cell line Raw 264.7 also inhibits RANKL-mediated osteoclastogenesis. LSD1-inhibitor-treated primary macrophage cells and LSD1 gene knockout Raw 264.7 cells failed to show actin ring formation. LSD1 inhibitors prevent the expression of RANKL-induced osteoclast-specific genes. They also downregulated the protein expression of osteoclast-related markers in osteoclastogeneses, such as Cathepsin K, c-Src, and NFATc1. Although LSD1 inhibitors were shown to reduce the in vitro demethylation activity of LSD1, they did not modulate the methylation of Histone 3 K4 and K9 during osteoclastogenesis. The ovariectomy (OVX)-induced osteoporosis model revealed that GSK2879552 slightly restores OVX-induced cortical bone loss. LSD1 can be employed as a positive regulator to promote osteoclast formation. Hence, inhibition of LSD1 activities is a potential target for preventing bone diseases characterized by excessive osteoclast activities.

Abstract ES13-3: Novel epigenomic targets in TNBC

Abstract Triple negative breast cancer is a heterogenous disease, characterized by a dearth of recurrent actionable genetic alterations. Epigenetic alterations have been implicated in the pathogenesis of triple negative breast cancer, as well as in the acquisition of drug resistance, which is a commonly observed phenomenon and persisting clinical challenge. An expanding array of tools for epigenomic characterization, together with novel selective inhibitors of epigenetic regulators are enabling new opportunities to identify and target these processes in triple negative breast cancer. Examples of recent and emerging therapeutic strategies using conventional therapies and epigenetic-targeted agents to exploit these vulnerabilities in triple negative breast cancer will be discussed. Citation Format: DW Cescon. Novel epigenomic targets in TNBC [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr ES13-3.

EXTH-71. FUNCTIONAL GENOMICS IDENTIFIES EPIGENETIC REGULATORS AS NOVEL THERAPEUTIC TARGETS FOR SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is among the most common malignant pediatric brain tumors. Among its four molecularly heterogeneous clinical variants, sonic hedgehog (SHH) subgroup comprises 30% of all MBs and is characterized by constitutive activation of the canonical SHH signaling pathway. Although Smoothened (Smo) inhibition has emerged as a promising therapeutic target for this tumor entity, primary or acquired resistance impedes its clinical efficacy. Thus, further insight into the molecular mechanisms underlying acquired resistance to Smo inhibition are urgently needed to overcome this challenge. Here, we performed a genome-wide CRISPR/Cas9 knockout screen in a murine and a human SHH-MB cell line, SMB21 and DAOY, respectively, in order to unravel tumor-specific genetic vulnerabilities. Our data provide functional evidence that SMB21 cells highly depend on key members of the SHH pathway such as Smo and Gli1 for their survival. In contrast, none of those positive regulators of SHH signaling scored in DAOY cells, suggesting that they are not a faithful human model of this tumor subtype. Of note, functional genomics identified SMB21-context specific essentialities beyond the SHH pathway that include epigenetic regulators such as Dnmt1, Smarca5 and Smarca4. Further in vitro pharmacological validations demonstrate that Dnmt1 inhibition is efficacious in clinically relevant concentrations in SHH-associated cell lines, both sensitive and resistant to Smo inhibition, suggesting novel therapeutic avenues for SHH-MB. By employing genome-scale knockout screens in murine cell lines faithfully recapitulating the biology of human SHH-MB, we aim to decipher synthetic lethal interactors for Dnmt1 inhibitors that could potentially serve as a combinatorial treatment approach for SHH-MB. Finally, genetic ablation and pharmacological inhibition of epigenetic regulators will be evaluated in in vivo mouse models of SHH-MB. Summarizing, our data highlight the potential of inhibitors of epigenetic regulators in SHH-MB sensitive, as well as resistant to Smo inhibition.

Epigenetic Regulation of Immunotherapy Response in Triple-Negative Breast Cancer.

Simple SummaryTriple-negative breast cancer (TNBC) outcomes are improving since the implementation of immunotherapy. However, objective response rates are still limited to a select group of patients. This is partly due to TNBC intrinsic immune evasive mechanisms and the lack of proper tumor microenvironment immune system activation. Dynamic epigenetic modifications contribute to immune surveillance and immune escape in cancer and can be reverted through epigenetic drugs. This review summarizes the epigenetic changes in TNBC cells and their contribution to the cancer cell–immunity cycle. Furthermore, it also describes how epigenetic drugs may provide novel biomarkers for immunotherapy and enhance the immune response. This manuscript lists the current clinical trials using epigenetic drugs alone or combined with either immune checkpoint inhibitors or small molecules.Triple-negative breast cancer (TNBC) is defined by the absence of estrogen receptor and progesterone receptor and human epidermal growth factor receptor 2 (HER2) overexpression. This malignancy, representing 15–20% of breast cancers, is a clinical challenge due to the lack of targeted treatments, higher intrinsic aggressiveness, and worse outcomes than other breast cancer subtypes. Immune checkpoint inhibitors have shown promising efficacy for early-stage and advanced TNBC, but this seems limited to a subgroup of patients. Understanding the underlying mechanisms that determine immunotherapy efficiency is essential to identifying which TNBC patients will respond to immunotherapy-based treatments and help to develop new therapeutic strategies. Emerging evidence supports that epigenetic alterations, including aberrant chromatin architecture conformation and the modulation of gene regulatory elements, are critical mechanisms for immune escape. These alterations are particularly interesting since they can be reverted through the inhibition of epigenetic regulators. For that reason, several recent studies suggest that the combination of epigenetic drugs and immunotherapeutic agents can boost anticancer immune responses. In this review, we focused on the contribution of epigenetics to the crosstalk between immune and cancer cells, its relevance on immunotherapy response in TNBC, and the potential benefits of combined treatments.

Targeting the epigenetic regulation of antitumour immunity.

Dysregulation of the epigenome drives aberrant transcriptional programmes that promote cancer onset and progression. Although defective gene regulation often affects oncogenic and tumour-suppressor networks, tumour immunogenicity and immune cells involved in antitumour responses may also be affected by epigenomic alterations. This could have important implications for the development and application of both epigenetic therapies and cancer immunotherapies, and combinations thereof. Here, we review the role of key aberrant epigenetic processes - DNA methylation and post-translational modification of histones - in tumour immunogenicity, as well as the effects of epigenetic modulation on antitumour immune cell function. We emphasize opportunities for small-molecule inhibitors of epigenetic regulators to enhance antitumour immune responses, and discuss the challenges of exploiting the complex interplay between cancer epigenetics and cancer immunology to develop treatment regimens combining epigenetic therapies with immunotherapies.

Epigenetic CRISPR Screens Identify Npm1 as a Therapeutic Vulnerability in Non-Small Cell Lung Cancer.

Despite advancements in treatment options, the overall cure and survival rates for non-small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 (Npm1) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Furthermore, KRAS-mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC. SIGNIFICANCE: Epigenome-wide CRISPR knockout screens identify NPM1 as a novel metabolic vulnerability and demonstrate that targeting NPM1 is a new therapeutic opportunity for patients with NSCLC.

In vivo efficacy of mutant IDH1 inhibitor HMS-101 and structural resolution of distinct binding site.

Mutations in isocitrate dehydrogenase 1 (IDH1) are found in 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. Here we report the biochemical characterization, in vivo antileukemic effects, structural binding and molecular mechanism of the inhibitor HMS-101, which inhibits the enzymatic activity of mutant IDH1 (IDH1mut). Treatment of IDH1mut primary AML cells reduced 2-hydroxyglutarate levels (2HG) and induced myeloid differentiation in vitro. Co-crystallization of HMS-101 and mutant IDH1 revealed that HMS-101 binds to the active site of IDH1mut in close proximity to the regulatory segment of the enzyme in contrast to other IDH1 inhibitors. HMS-101 also suppressed 2HG production, induced cellular differentiation and prolonged survival in a syngeneic mutant IDH1 mouse model and a patient-derived human AML xenograft model in vivo. Cells treated with HMS-101 showed a marked upregulation of the differentiation-associated transcription factors CEBPA and PU.1, and a decrease in cell cycle regulator cyclin A2. In addition, the compound attenuated histone hypermethylation. Together, HMS-101 is a unique inhibitor that binds to the active site of IDH1mut directly and is active in IDH1mut preclinical models.

Signaling Pathways of Mutant IDH1 Independent of R-2-Hydroxyglutarate

#Michael Heuser and Anuhar Chaturvedi share senior authorshipBackground: Isocitrate dehydrogenase-1 (IDH1) is mutated in about 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, thereby linking metabolism to tumorigenesis. We recently reported that at comparable intracellular R-2HG levels, mice receiving transplants of IDH1 mutant cells died significantly earlier than R-2HG treated mice in the context of HOXA9 overexpression. This suggests oncogenic functions of mutant IDH1 beyond R-2HG production. We employed a splice variant of mutated IDH1 that does not produce R-2HG (IDH1mutantΔ7) to decipher R-2HG independent signaling pathways that may contribute towards leukemogenesis.Methods: Bone marrow cells from mice were immortalized with HoxA9, and IDH1wildtype (IDH1wt), IDH1mutant (IDH1mut), IDH1wildtypeΔ7 (IDH1wtΔ7) and IDH1mutΔ7, were constitutively expressed and the leukemogenic potential was evaluated in vivo. Intracellular R-2HG was measured by enantiomer-specific quantification. Deletion of exon 7 from IDH1mut leads to a frameshift that creates a premature stop codon in the 9th exon, finally producing a 119 amino acids truncated protein, IDH1mutΔ7. This splice variant does not produce increased levels of R-2HG. The signaling pathways were explored by immunoblotting and immunofluorescence.Results: Mice receiving cells with IDH1mutΔ7 had the same short latency to leukemia as mice receiving cells with full-length mutant IDH1, while IDH1wt and IDH1wtΔ7 cells died with significantly longer latency. The WBC count increased over time in IDH1mutΔ7 mice similar to IDH1mut mice, whereas WBC counts in IDH1wtΔ7 mice remained normal. IDH1mutΔ7 mice died from monocytic leukemia that was phenotypically and morphologically indistinguishable from IDH1mut mice. HoxA9 IDH1mutΔ7 cells were readily transplantable into secondary recipients. During in vivo cell cycle analysis, we observed that the proportion of cells in S/G2/M phases was significantly higher in bone marrow cells transduced with IDH1mut or IDH1mutΔ7 when compared to cells transduced with IDH1wt or CTL. These data suggest that mutant IDH1 enhances myeloproliferation even in the absence of R-2HG.To identify R-2HG independent signaling pathways mediated by the mutant IDH1 protein, we first analyzed the gene expression of important regulators of cell cycle, differentiation, cell signaling and transcription by quantitative RT-PCR. Several genes (Ccnd1, Slc2a, Hdac3, Tgif2,and c-myc) were upregulated in IDH1mut and IDH1mutΔ7 cells compared to IDH1wt cells. Interestingly, we found a specific up-regulation of Ctnnb1 and Nfkb genes in IDH1mutΔ7 cells over both IDH1mut and IDH1wt cells. We next validated our mRNA expression results by immunoblotting and found that NFKB and ERK signaling were upregulated in both IDH1mut and IDH1mutΔ7 compared to IDH1wt and IDH1wtΔ7 cells. Interestingly, the protein level of β-catenin, STAT3 and STAT5 were many fold higher in IDH1mutΔ7 compared to IDH1mut and IDH1wt cells. β-catenin is known to be transactivated via c-Src, which is phosphorylated by EGFR to promote β-catenin nuclear localization and signaling. We traced this pathway for its relevance in our cells and found that IDH1mutΔ7 cells indeed showed higher levels of both EGFR and c-Src phosphorylation compared to IDH1mut cells. We performed immunofluorescence and cellular fractionation for β-catenin and found it to be partially localized in the nucleus in IDH1mutΔ7 but not in IDH1mut cells. We also observed an up-regulated STAT3 phosphorylation in IDH1mutΔ7 cells over IDH1mut.Conclusions: In summary, mutant IDH1 activates ERK and NFKB signaling, which is attributed to both R-2HG dependent and independent mechanisms of leukemogenesis. Interestingly, IDH1mutΔ7 employs R-2HG independent EGFR/β-catenin and JAK/STAT signaling for oncogenesis. This R-2HG-independent leukemogenesis reveals a novel signaling dynamic of IDH1mut which should be evaluated for its therapeutic potential. DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Astellas: Research Funding; Karyopharm: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; StemLine Therapeutics: Consultancy; Bayer Pharma AG: Consultancy, Research Funding; Sunesis: Research Funding; BergenBio: Research Funding; Tetralogic: Research Funding; Daiichi Sankyo: Research Funding.

Abstract 5867: Identification of novel inhibitors of the PAX3-FOXO1 oncogenic driver in rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, representing 8% of all childhood tumors. The most aggressive subtype of RMS is associated with chromosomal translocations creating a fusion transcription factor, most commonly involving PAX3 and FOXO1 (fusion-positive (FP)-RMS). Our group has recently comprehensively characterized the genetic and epigenetic landscape of FP-RMS. We found that FP-RMS has a strikingly low somatic mutational burden and that the PAX3-FOXO1 fusion gene is the primary oncogenic driver. Specifically, we found that PAX3-FOXO1 reprograms the cis-regulatory epigenetic landscape by inducing de novo super enhancers in collaboration with the bromodomain and extra-terminal domain (BET) protein family member BRD4, and the master transcription factors MYOG, MYOD1, and MYCN. We therefore hypothesized that FP-RMS would be vulnerable to disruption of the PAX3-FOXO1 super enhancer oncogenic circuitry. Method and Results: To test this, we developed a novel readout assay to simultaneously monitor PAX3-FOXO1 super enhancer and general transcriptional activity as well as cell viability, and screened against a 62,643 small-molecule library at a concentration of 10 µM. We found a total of 573 compounds with selective ALK super enhancer inhibition, which were selected for dose response assay with four 10-fold dilutions ranging from 20 μM to 2 nM. Further prioritization using a systematic computational model reduced this number to 64. Among the compounds with known activities, the multi-kinase PKC-412/midostaurin was previously identified in screens for PAX3-FOXO1 inhibitors. Other known compounds included topoisomerase I and topoisomerase II inhibitors, where both enzymes have recently been reported to play important roles in BRD4-facilitated DNA dynamics involving super enhancers and the transcriptional machinery. Among this list we identified 33 novel compounds of unknown mechanism of action. We therefore performed RNA-seq analysis to determine the transcriptional impact of the effect of these compounds. Pathway, gene set enrichment and Connectivity Map analyses of RNA-seq expression signatures implicate several of the novel compounds as inhibitors of epigenetic regulators, including inhibitors of BRD4, histone deacetylases, histone demethylases, histone methyltransferases, and chromodomain proteins. Conclusions: Our systematic approach identified several small molecules that specifically disrupt the activity of the PAX3-FOXO1 fusion oncogene in RMS. Further characterization of the specificity and selectivity of the lead compounds is ongoing, and exact mechanism of action is currently being elucidated. Our ultimate goal is to develop a clinically effective precision therapeutic targeting the previously “undruggable” PAX3-FOXO1 oncogenic driver of aggressive FP-RMS. Citation Format: Robert G. Hawley, Girma M. Woldemichael, Berkley E. Gryder, Young Song, Sivasish Sindiri, Vineela Gangalapudi, Jun S. Wei, James B. McMahon, Jack F. Shern, Javed Khan. Identification of novel inhibitors of the PAX3-FOXO1 oncogenic driver in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5867.

Epigenetic regulation of interleukin-8 expression by class I HDAC and CBP in ovarian cancer cells.

Although inhibitors of epigenetic regulators have been effective in the treatment of cutaneous T cell lymphoma (CTCL) and other hematopoietic malignancies, they have been less effective in solid tumors, including ovarian cancer (OC). We have previously shown that inhibition of histone deacetylase (HDAC) activity induces expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (CXCL8, IL-8) in OC cells, resulting in their increased survival and proliferation. Here, we show that in addition to ovarian cancer SKOV3, OVCAR3, and CAOV3 cells, HDAC inhibition induces the CXCL8 expression in HeLa cells, but not in CTCL Hut-78 cells. In OC cells, the CXCL8 expression is induced by pharmacological inhibition of class I HDACs. Interestingly, while an individual suppression of HDAC1, HDAC2, or HDAC3 by corresponding siRNAs inhibits the CXCL8 expression, their simultaneous suppression induces the CXCL8 expression. The induced CXCL8 expression in OC cells is dependent on histone acetyltransferase (HAT) activity of CREB-binding protein (CBP), but not p300, and is associated with HAT-dependent p65 recruitment to CXCL8 promoter. Together, our results show that the CXCL8 expression in OC cells is induced by combined inhibition of HDAC1, -2, and -3, and silenced by suppression of HAT activity of CBP. In addition, our data indicate that the induced CXCL8 expression may be responsible for the limited effectiveness of HDAC inhibitors in OC and perhaps other solid cancers characterized by CXCL8 overexpression, and suggest that targeting class I HDACs and CBP may provide novel combination strategies by limiting the induced CXCL8 expression.

Quantitative proteomic analysis of EZH2 inhibition in acute myeloid leukemia reveals the targets and pathways that precede the induction of cell death.

Chromosomal translocation of the mixed lineage leukemia (MLL) locus generates fusion proteins that drive acute myeloid leukemia (AML) resulting in atypical histone methyltransferase activity and alterations in the epigenetic regulation of gene expression. Targeting histone regulators, such as Enhancer of Zeste Homologue 2 (EZH2), has shown promise in AML. Profiling differential protein expression following inhibition of epigenetic regulators in AML may help to identify novel targets for therapeutics. Murine models of AML combined with quantitative SILAC analysis were used to identify differentially expressed proteins following inhibition of EZH2 activity using 3-Deazaneplanocin A (DZnep). Western blotting and flow cytometry were used to validate a subset of differentially expressed proteins. Gene set analysis was used to determine changes to reported EZH2 target genes. Our quantitative proteomic analysis and subsequent validation of protein changes identified that epigenetic therapy leads to cell death preceded by the induction of differentiation with concurrent p53 up-regulation and cell cycle arrest. Gene set analysis revealed a specific subset of EZH2 target genes that were regulated by DZnep in AML. These discoveries highlight how this new class of drugs affects AML cell biology and cell survival, and may help identify novel targets and strategies to increase treatment efficacy.

Pan-Mutant-IDH1 Inhibitor Bay-1436032 Is Highly Effective Against Human IDH1 Mutant Acute Myeloid Leukemia In Vivo

Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are frequently found in patients with acute myeloid leukemia (AML) and several other tumors. Mutant IDH1 produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, and leads to a block in differentiation to promote tumorigenesis.Methods: We developed a novel, highly active oral pan-IDH1 inhibitor, BAY-1436032, for clinical evaluation. Its inhibitory potency was evaluated in primary human AML cells in vitro for the five major IDH1R132 mutation types and in two patient derived AML xenograft (PDX) models in vivo, in which BAY-1436032 cleared leukemic blasts in peripheral blood and prolonged survival by induction of differentiation and inhibition of leukemia stem cell self-renewal.Results: R-2HG production by mutant IDH1 was effectively inhibited in patient derived AML cells with all reported IDH1R132 mutations ex vivo by BAY-1436032 with an IC50 between 3 to 16 nM. AML cells cultured ex vivo showed morphologic differentiation and marked upregulation of the myeloid differentiation markers CD14 and CD15.For in vivo experiments, human AML cells from two patients were transplanted into sublethally irradiated NSG mice. After stable engraftment at 17 (PDX1) or 90 (PDX2) days post transplantation, mice were treated with BAY-1436032 orally every day at a dose of 150 mg/kg or vehicle for 100-150 days (n=10 per group). The R/S-2HG ratio in serum was reduced to near normal levels by BAY-1436032. Leukemic cell counts in peripheral blood constantly increased in control mice, while leukemic cells declined from day 30 of BAY-1436032 treatment onwards with morphologic and immunophenotypic evidence of differentiation (Figure). Importantly, all BAY-1436032 treated PDX1 mice survived until the end of treatment at 150 days. In contrast, vehicle-treated mice died with a median latency of 91 days (range 70-95, P<.001). In an independent second model (PDX2) 6 of 10 BAY-1436032 treated mice survived until the end of treatment at day 100 with a median of 15% leukemic cells in peripheral blood, while all vehicle-treated mice suffered from high leukemic burden and died from leukemia with a median survival of 62 days (P=.014). Early mortality was increased with 4 mice dying in the BAY-1436032 group reminiscent of clinical differentiation syndrome in AML patients treated with the IDH1 inhibitor AG-120.To assess the effect of BAY-1436032 on leukemic stem cell self-renewal we treated PDX1 mice with 150 mg/kg BAY-1436032 or vehicle for 4 weeks and performed a limiting dilution transplantation experiment in secondary recipient mice. LSC frequency was 100-fold lower in BAY-1436032 treated compared to control mice. Gene expression profiling showed that stemness associated genes were downregulated, while genes associated with myeloid differentiation like PU.1 and CEBPA were upregulated upon treatment with BAY-1436032. In addition, cell cycle progression was slowed and E2F transcription factors concomitantly inhibited. In accordance with gene expression profiling results, methylation of the PU.1 promoter decreased, while E2F1 promoter methylation increased upon treatment with BAY-1436032. Finally, histone trimethylation levels at residues H3K4, H3K9, H3K27, and H3K36 decreased in both IDH1R132C and IDH1R132H mutant AML cells but not in IDH1 wildtype cells upon BAY-1436032 treatment.Conclusion: In summary, the novel oral pan-mutant IDH1 inhibitor BAY-1436032 is active against all IDH1R132 mutation types and shows strong anti-leukemic activity in two independent AML PDX mouse models. Clinical development is ongoing with a first in man study with BAY-1436032 in IDH1 mutant solid tumors.* M. Heuser and L. Herbst contributed equally to this article#A. Krämer and A. Chaturvedi share senior authorship [Display omitted] DisclosuresHeuser:Tetralogic: Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding; Celgene: Honoraria; Novartis: Consultancy, Research Funding; Pfizer: Research Funding. Pusch:German Cancer Research Center: Patents & Royalties: WO2013/127997A1. Kaulfuss:Bayer Pharma AG: Employment. Panknin:Bayer Pharma AG: Employment. Zimmermann:Bayer Pharma AG: Employment, Patents & Royalties: WO2015/121210 . Toschi:Bayer Pharma AG: Employment. Neuhaus:Bayer Pharma AG: Employment, Patents & Royalties: WO2015/121210. Haegebarth:Bayer Pharma AG: Employment, Equity Ownership. Rehwinkel:Bayer Pharma AG: Employment, Equity Ownership, Patents & Royalties: WO2015/121210. Hess-Stumpp:Bayer Pharma AG: Employment. Bauser:Bayer Pharma AG: Employment. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. von Deimling:German Cancer Research Center: Patents & Royalties: IDH1R132H mutant specific antibody H09; BRAF V600E mutant specific antibody VE1; BAY-1436032 patent.

The epigenetic regulators CBP and p300 facilitate leukemogenesis and represent therapeutic targets in acute myeloid leukemia.

Growing evidence links abnormal epigenetic control to the development of hematological malignancies. Accordingly, inhibition of epigenetic regulators is emerging as a promising therapeutic strategy. The acetylation status of lysine residues in histone tails is one of a number of epigenetic post-translational modifications that alter DNA-templated processes, such as transcription, to facilitate malignant transformation. Although histone deacetylases are already being clinically targeted, the role of histone lysine acetyltransferases (KAT) in malignancy is less well characterized. We chose to study this question in the context of acute myeloid leukemia (AML), where, using in vitro and in vivo genetic ablation and knockdown experiments in murine models, we demonstrate a role for the epigenetic regulators CBP and p300 in the induction and maintenance of AML. Furthermore, using selective small molecule inhibitors of their lysine acetyltransferase activity, we validate CBP/p300 as therapeutic targets in vitro across a wide range of human AML subtypes. We proceed to show that growth retardation occurs through the induction of transcriptional changes that induce apoptosis and cell-cycle arrest in leukemia cells and finally demonstrate the efficacy of the KAT inhibitors in decreasing clonogenic growth of primary AML patient samples. Taken together, these data suggest that CBP/p300 are promising therapeutic targets across multiple subtypes in AML.

A Novel Inhibitor of Mutant IDH1 Induces Differentiation in Vivo and Prolongs Survival in a Mouse Model of Leukemia

Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in patients with glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, cholangiocellular carcinoma and chondrosarcoma. Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. We recently established an in vivo mouse model and investigated the function of mutant IDH1. By computational drug screening, we identified an inhibitor of mutant IDH1 (HMS-101), which inhibits mutant IDH1 cell proliferation, decreases R2HG levels in vitro, and efficiently blocks colony formation of AML cells from IDH1 mutated patients but not of normal CD34+ bone marrow cells. In the present study we investigated the effect of the inhibitor in our IDH1/HoxA9-induced mouse model of leukemia in vivo.To identify the maximally tolerated dose of HMS-101, we treated normal C57BL/6 mice with variable doses of HMS-101 for 9 days and measured the serum concentration. Mice receiving 0.5 mg and 1mg intraperitoneally once a day tolerated the drug well with mean plasma concentrations of 0.1 to 0.3 µM. To evaluate the effect of HMS-101 in the IDH1 mouse model, we transduced IDH1 R132C in HoxA9-immortalized murine bone marrow cells. Sorted transgene positive cells were then transplanted into lethally irradiated mice. After 5 days of transplantation, mice were treated with HMS-101 intraperitoneally for 5 days/week. The R/S-2HG ratio in serum was reduced 3-fold in HMS-101 treated mice after 8 weeks of treatment compared to control treated mice. HMS-101 or PBS treated mice had similar levels of transduced leukemic cells in peripheral blood at 2 and 6 weeks after transplantation. However, from week 6 to week 15 leukemic cells in peripheral blood decreased from 76% to 58, 63% to 29%, 67% to 7%, and 74% to 38% in 4/6 mice treated with HMS-101. In one mouse the percentage of leukemic cells was constant, and in one mouse it increased from week 6 to week 15 after transplantation. Leukemic cells increased constantly in peripheral blood until death in control treated mice. While the control cohort developed severe leukocytosis, anemia and thrombocytopenia around 8 to 10 weeks post transplantation, mice treated with HMS-101 still had normal WBC, RBC and platelet counts at 15 weeks after transplantation. Moreover, the HMS-101 treated mice had significantly more differentiated Gr1+CD11b+ cells in peripheral blood than control mice at 6 weeks and 15 weeks after transplantation and at death (P=.01). Morphologic evaluation of blood cells at 15 weeks or death from HMS-101 treated mice revealed a high proportion of mature neutrophils that were GFP positive and thus derived from IDH1 transduced cells, whereas control treated mice had monocytic morphology with a high proportion of immature cells. Importantly, HMS-101 treated mice survived significantly longer with a median latency of 87 days (range 80-118), whereas PBS-treated mice died with a median latency of 66 days (range 64-69) after transplantation (P<.001). Of note, HMS-101 was found to be specific for mutant IDH1, as mutant IDH2 cells were not preferentially inhibited over IDH2 wildtype cells in vitro.This data demonstrates that HMS-101 specifically inhibits R2HG-production of mutant IDH1 in vivo, inhibits proliferation, induces differentiation in leukemic cells, and thus prolongs survival of IDH1mutant leukemic mice. Therefore, HMS-101 - a novel inhibitor of mutant IDH1 - shows promising activity in vivo and warrants further development towards clinical use in IDH1 mutated patients. DisclosuresChaturvedi:Hannover Medical School: Patents & Royalties. Preller:Hannover Medical School: Patents & Royalties. Heuser:Hannover Medical School: Patents & Royalties.

The Metabolite R-2-Hydroxyglutarate (R2HG) Collaborates with HoxA9 to Induce Monocytic Leukemia

Mutations in the metabolic enzymes IDH1 and IDH2 are frequently found in several tumors including glioma and acute myeloid leukemia (AML). Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. However, it is unknown whether R2HG alone is sufficient to recapitulate the biologic effects of mutant IDH1 in vivo. Recently, we have shown that IDH1mut cooperates with HoxA9 and induces a monocytic leukemia in mice. In order to evaluate the effects of R2HG independently of the mutated IDH1 protein and to determine whether the effects are specific to the R-enantiomer of 2HG, we treated mice transplanted with HoxA9 immortalised bone marrow cells with R2HG, S-2-hydroxyglutarate (S2HG), alpha-ketoglutarate (aKG) and phosphate buffered saline (PBS). The mice in the metabolite cohorts received an intraperitoneal dose of 1 mg per day.Mice treated with R2HG had higher engraftment levels at 16 and 20 weeks post transplantation than the mice treated with S2HG, αKG and PBS respectively (P<.01). High WBC counts (70±16 /nl) and lower platelet counts than in control mice were observed in the cohort receiving R2HG after 16 to 20 weeks of treatment, while the S2HG, αKG and PBS cohorts had normal blood counts even at 20 weeks (P<.05). Peripheral blood from R2HG treated mice revealed significantly more immature Mac1+Gr1- and less mature Mac1+Gr1+ cells at 12 and 16 weeks after treatment than S2HG, αKG and PBS treated mice (P<.001). In addition, the R2HG treated mice died with a median latency of 137 days post transplantation from monocytic leukemia, while mice treated with S2HG, αKG and PBS died with a median latency of 223, 202 and 184 days respectively (P<.001). Further, in order to assess whether R2HG alone was sufficient as a single hit to induce myeloproliferation, normal C57BL/6 mice (without HoxA9) were treated with R2HG, S2HG and PBS for eight months. No differences were observed for survival, blood counts, immunophenotype and frequencies of progenitor cells (lin-ckit+sca1+, CMP, GMP and MEP) between treatment groups and control. This data shows that the metabolite R2HG like the IDH1 mutant protein cooperates with HoxA9 to induce monocytic leukemia.We next compared mice receiving transplants of HoxA9+IDH1mut cells with mice receiving HoxA9 cells that were then treated with R2HG. Both cohorts developed monocytic leukemia, albeit with different kinetics. The Hoxa9+IDH1mut mice died with a median latency of 83 days while the R2HG cohort died with a median latency of 137 days post transplantation (P<0.001). Also, while the former cohort developed severe leukocytosis, anemia and thrombocytopenia at 12 weeks, the R2HG treated mice had high WBC counts and lower platelet counts than control mice at 16 to 20 weeks after treatment. The faster disease kinetics in IDH1mut mice could be attributed to a significantly lower proportion of cells in G0/G1 and higher proportion of cells in S phase when compared to cells from mice treated with R2HG at 9 weeks after transplantation (P<.001). This resulted from a marked downregulation of cyclin-dependent kinase inhibitors (Cdkn) 1A (p21), 1B (p27), 2A (p16), and 2B (p15) in HoxA9+IDH1mut cells as compared to HoxA9 cells treated with R2HG or PBS.In order to rule out an influence of differential R2HG levels on the disease progression between the two cohorts, levels of R2HG were quantified. IDH1mut expressing and R2HG treated bone marrow cells from mice had similarly high ratios of R2HG/S2HG. The quantified R2HG levels were also comparable to that of primary AML patient cells harbouring mutated IDH1. In unsupervised hierarchical clustering mutated cells clustered together with R2HG and separated from PBS treated mice. 69 of the top 100 enriched Gene Ontology gene sets of HoxA9+IDH1mut were also found in HoxA9+R2HG, suggesting largely redundant but also non-overlapping functions of the mutant IDH1 protein and the oncometabolite R2HG.In summary, we show that R2HG, similar to the mutant IDH1 protein, promotes leukemogenesis in cooperation with HoxA9, although with delayed kinetics. Our data proves that R2HG acts as an oncometabolite in vivo in a murine model of leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Targeting epigenetic regulators for cancer therapy

Human gene expression patterns are controlled and coordinated by the activity of a diverse array of epigenetic regulators, including histone methyltransferases, acetyltransferases, and chromatin remodelers. Deregulation of these epigenetic pathways can lead to genome-wide changes in gene expression, with serious disease consequences. In recent years, research has suggested that cross talk between genomic (i.e., for example, mutations, translocations) and epigenomic factors may drive the etiology of both hematologic malignancies and solid tumors. Current work in translational research seeks to identify epigenetic regulators whose aberrant activity contributes to oncogenesis, including the histone methyltransferases DOT1L and EZH2 and the bromodomain-containing BET family, and to develop drugs that inhibit the aberrant activity of these regulators. Preclinical and clinical studies using small-molecule inhibitors of epigenetic regulators have underscored their value for therapeutic intervention, and these inhibitors can also be used to drive further studies into dissecting the functions of epigenetic factors in normal and cancer cells.

Mutant IDH1 promotes leukemogenesis in vivo and can be specifically targeted in human AML

AbstractMutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, and chondrosarcoma patients. Mutant IDH produces 2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators. We investigated the role of mutant IDH1 using the mouse transplantation assay. Mutant IDH1 alone did not transform hematopoietic cells during 5 months of observation. However, mutant IDH1 greatly accelerated onset of myeloproliferative disease–like myeloid leukemia in mice in cooperation with HoxA9 with a mean latency of 83 days compared with cells expressing HoxA9 and wild-type IDH1 or a control vector (167 and 210 days, respectively, P = .001). Mutant IDH1 accelerated cell-cycle transition through repression of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, and activated mitogen-activated protein kinase signaling. By computational screening, we identified an inhibitor of mutant IDH1, which inhibited mutant IDH1 cells and lowered 2HG levels in vitro, and efficiently blocked colony formation of AML cells from IDH1-mutated patients but not of normal CD34+ bone marrow cells. These data demonstrate that mutant IDH1 has oncogenic activity in vivo and suggest that it is a promising therapeutic target in human AML cells.

Mutated IDH1 Has 2-Hydroxyglutarate-Independent Functions in Leukemogenesis

AbstractAbstract 770Mutations in the metabolic enzymes IDH1 and IDH2 are frequently found in glioma and AML patients. Mutant IDH produces R-2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. However, it is unknown whether R-2HG is required for transformation. To investigate the function of mutated IDH1 we established an in vivo mouse model, which allowed us to study R-2HG-dependent and –independent functions of mutated IDH1.We cloned wildtype IDH1 (IDH1wt) and mutated IDH1 (IDH1mut, R132C) from AML and MDS patients and identified a mutated splice variant that lacked exon 7 (IDH1mutΔ7) in several IDH1 mutated patients. To evaluate whether the mutated proteins produce the oncometabolite R-2HG, we expressed IDH1mut, IDH1mutΔ7, and IDH1wt in immortalized murine bone marrow cells and measured intracellular levels of 2HG by enantiomer-specific quantification. IDH1mut expressing cells produced high levels of R-2HG, but not IDH1 mutated cells that lacked exon 7 of IDH1, control-vector transduced cells (CTL), or IDH1 wildtype cells (P<.001). To functionally analyze mutated IDH1, we transduced IDH1wt, IDH1wtΔ7, IDH1mut, IDH1mutΔ7, or empty vector CTL in HoxA9-immortalized murine bone marrow cells and transplanted sorted cells in lethally irradiated mice. Mice receiving transplants with IDH1mut and IDH1mutΔ7 transduced cells had higher engraftment levels at 4, 8, and 12 weeks after transplantation (P<.001) than IDH1wt, IDH1wtΔ7, and CTL mice, and developed severe leukocytosis, anemia and thrombocytopenia, whereas IDH1wt, IDH1wtΔ7, and CTL mice had normal blood counts at 12 weeks. Mice with mutated IDH1, whether producing 2HG (IDH1mut) or not (IDH1mutΔ7), died with a median latency of 83 and 80 days after transplantation, respectively, whereas IDH1wt and CTL mice survived for a median of 167 and 210 days, respectively, and IDH1wtΔ7 mice were alive at 200 days (pooled data from 3 independently transduced cell populations that were transplanted at 3 different time points, P<.001). IDH1mut and IDH1mutΔ7 mice died with a myeloproliferative-like disease with high white blood cell counts, large spleen, anemia and thrombocytopenia. Bone marrow cells from moribund mice were readily transplantable in secondary animals, and rapidly induced disease. These data demonstrate that myeloproliferation is accelerated by an alternatively spliced mutant IDH1 independent of the metabolite R-2HG.In vivo cell cycle analysis showed a significantly higher proportion of cells in S/G2/M phase in bone marrow cells transduced with IDH1mut or IDH1mutΔ7 when compared to cells transduced with IDH1wt or CTL. Also, cyclin-dependent kinase inhibitors (Cdkn) 1A (p21), 1B (p27), 2A (p16), and 2B (p15) were markedly downregulated in IDH1mut and IDH1mutΔ7 cells when compared to IDH1wt cells. We next investigated, whether the promoters of the repressed Cdk inhibitors were hypermethylated. The CpG island in the promoter of Cdkn2a and Cdkn2b showed low levels of DNA methylation in IDH1wt, IDH1mut, IDH1mutΔ7 and CTL cells (0.6% to 4.3% average methylation), suggesting that Cdk inhibitors are repressed in cells with mutated IDH1 independently of DNA methylation. Gene set enrichment analysis from microarray data of transduced bone marrow cells showed that genes related to MAP-kinase signalling were highly enriched in IDH1mut and IDHmutΔ7 cells compared to IDH1wt or CTL transduced cells. By Western blot we found that pERK was highly upregulated in IDH1mut and IDHmutΔ7 cells compared to IDH1wt or CTL cells. Pharmacologic inhibition of transduced cells in vitro showed that IDH1mut and IDH1mutΔ7 cells were resistant to inhibition with the MEK1/2 inhibitor UO126, suggesting that MAP-kinase signalling is a relevant target in IDH1 mutated AML.In summary, we show that an alternatively spliced isoform of mutated IDH1 promotes leukemogenesis independently of R-2HG in a mouse model of transformed hematopoietic cells with the same kinetics as mutated full-length IDH1 through transcriptional repression of cyclin-dependent-kinase inhibitors p15 and p16 and activation of MAP-kinase signalling. Our mouse model of mutated IDH1 represents a powerful tool to test the efficacy of newly developed drugs targeting mutated IDH1 in a 2-HG-dependent and –independent manner. Disclosures:No relevant conflicts of interest to declare.